Cervical Cancer Cell Lines SiHa Research Articles

HPV16-miRNAs exert oncogenic effects through enhancers in human cervical cancer

BackgroundCervical cancer is a human papillomavirus (HPV)-related disease. HPV type 16 (HPV16), which is the predominant cause of cervical cancer, can encode miRNAs (HPV16-miRNAs). However, the role of HPV16-miRNAs in the pathogenesis of cervical cancer remains unclear.MethodsHuman cervical cancer cell lines SiHa (HPV16-positive) and C33A (HPV-negative), and cervical cancer tissues were collected to investigate the expression levels of two HPV16-miRNAs (HPV16-miR-H1 and HPV16-miR-H6). The overexpression and knockdown of HPV16-miR-H1 and HPV16-miR-H6 were performed using the lentiviral vector system and miRNA inhibitors, respectively. RNA-sequencing (RNA-seq) analysis and H3K27ac chromatin immunoprecipitation and sequencing (CHIP-seq) experiments were utilized to explore the roles of HPV16-miR-H1 and HPV16-miR-H6 facilitated by enhancers. CCK8, EdU, transwell, and wound healing assays were performed to verify the effects of HPV16-miR-H1 and HPV16-miR-H6 on cell proliferation and migration.ResultsHPV16-miR-H1 and HPV16-miR-H6 were highly expressed in both SiHa cells and tissue samples from HPV16-positive cervical cancer patients. RNA-seq analysis showed that HPV16-miR-H1 and HPV16-miR-H6 induced the upregulation of numerous tumor progression-associated genes. H3K27ac CHIP-seq experiments further revealed that HPV16-miR-H1 and HPV16-miR-H6 modulated the expression of critical genes by regulating their enhancer activity. The functional study demonstrated that HPV16-miR-H1 and HPV16-miR-H6 increased the migratory capacity of SiHa cells.ConclusionsOur data shed light on the role of HPV16-encoded miRNAs in cervical cancer, particularly emphasizing their involvement in the miRNA-enhancer-target gene system. This novel regulatory mechanism of HPV16-miRNAs provides new insights and approaches for the development of therapeutic strategies by targeting HPV16-positive cervical cancer.

Design, synthesis and molecular docking of 5-fluoro indole derivatives as inhibitors of PI3K/Akt signalling pathway in cervical cancer

The PI3K/Akt signalling pathway promotes variety of cellular processes and the inhibition of PI3K/Akt signalling pathway could lead to decrease in tumour growth effectively in cancer cells. AD412, an indole derivative, is a potent immunosuppressive agent which also reported as an anticancer agent through significant inhibition of PI3K/Akt signalling pathway. In this current work, we designed and synthesized the two diverse lead series of 5-fluoro indole derivatives (6a-l and 11a-l) by specific structural modifications of AD412. In total, 24 new derivatives were evaluated for their antiproliferative activity against two cervical cancer cell lines (HeLa and SiHa) and a normal cell line (HEK 293). Among them, 6e exhibited excellent antiproliferative activity against HeLa and SiHa cells with IC50 values of 9.366 and 8.475 µM respectively, as well displayed a low toxicity profile. Further, 6e inhibited the migration and invasion of HeLa cells in a dose-dependent manner by affecting the synthesis of DNA. Moreover, the Western blot analysis revealed that 6e could inhibit cervical cancer progression by downregulating the PI3K-p85 and phosphorylation of Akt in Hela cells. In vitro mechanism studies demonstrated that 6e could significantly increase apoptosis in HeLa cells by upregulating the expression of proapoptosis related protein Bax. The binding mechanism and the activity profile of 5-fluoro indole derivatives were validated by employing molecular docking studies against the active sites of Akt and PI3K enzymes. In addition, in silico ADME and pharmacokinetic parameters prediction of compound 6e resulted in good oral bioavailability. Therefore, compound 6e could be a lead compound for further development of PI3K/Akt inhibitors and anticancer agents.

Açaí (Euterpe oleracea Mart.) Seed Oil and Its Nanoemulsion: Chemical Characterisation, Toxicity Evaluation, Antioxidant and Anticancer Activities.

This study explores a nanoemulsion formulated with açaí seed oil, known for its rich fatty acid composition and diverse biological activities. This study aimed to characterise a nanoemulsion formulated with açaí seed oil and explore its cytotoxic effects on HeLa and SiHa cervical cancer cell lines, alongside assessing its antioxidant and toxicity properties both in vitro and in vivo. Extracted from fruits sourced in Brazil, the oil underwent thorough chemical characterization using gas chromatography-mass spectrometry. The resulting nanoemulsion was prepared and evaluated for stability, particle size, and antioxidant properties. The nanoemulsion exhibited translucency, fluidity, and stability post centrifugation and temperature tests, with a droplet size of 238.37, PDI -9.59, pH 7, and turbidity 0.267. In vitro assessments on cervical cancer cell lines revealed antitumour effects, including inhibition of cell proliferation, migration, and colony formation. Toxicity tests conducted in cell cultures and female Swiss mice demonstrated no adverse effects of both açaí seed oil and nanoemulsion. Overall, açaí seed oil, particularly when formulated into a nanoemulsion, presents potential for cancer treatment due to its bioactive properties and safety profile.

Design, synthesis, and biological evaluation of novel benzimidazole derivatives as anti-cervical cancer agents through PI3K/Akt/mTOR pathway and tubulin inhibition

Phosphatidylinositol 3-kinase (PI3K) is one of the most attractive therapeutic targets for cervical cancer treatment. In this study, we designed and synthesized a series of benzimidazole derivatives and evaluated their anti-cervical cancer activity. Compound 4r exhibited strong antiproliferative activity in different cervical cancer cell lines HeLa, SiHa and Ca Ski, and relative lower cytotoxicity to normal hepatic and renal cell lines LO2 and HEK-293t (IC50 values were at 21.08 μM and 23.96 μM respectively). Its IC50 value was at 3.38 μM to the SiHa cells. Further mechanistic studies revealed that 4r induced apoptosis, arrested cell cycle in G2/M phase, suppressed PI3K/Akt/mTOR pathway and inhibit the polymerization of tubulin. Molecular docking study suggested that 4r formed key H-bonds action with PI3Kα (PDB ID:8EXU) and tubulin (PDB ID:1SA0). Zebrafish acute toxicity experiments showed that high concentrations of 4r did not cause death or malformation of zebrafish embryos. All these results demonstrated that 4r would be a promising lead candidate for further development of novel PI3K and tubulin dual inhibitors in cervical cancer treatment.

Abstract 1114: Evaluating strategies to harness metabolic vulnerabilities in cervical

Abstract Tumor cells frequently alter their metabolism to generate biomolecular intermediates for tumor maintenance and proliferation. Previous reports from Dr. Julie Schwarz’s laboratory have shown that cervical cancer cells with altered AKT/mTOR pathways show increased reliance on glucose and exogenous glutamine. The laboratory has introduced PIK3CA E545K mutation and PTEN null mutation in cervical cancer cell line SiHa. Further, to evaluate the role of glutamine metabolism in these genotypes, these cells were cultured in sequentially lower levels of glutamine until these cells were able to proliferate without exogenous glutamine supplementation. These isogenic long-term glutamine-deprived (QD) cells allow us to tease out the role of these specific mutations in cellular metabolism. In our study, long-term glutamine deprivation inhibits the proliferation of rapidly proliferating PI3K-AKT-altered cell lines. Even though glutamine is required for GSH synthesis, which quenches reactive oxygen species, QD cells were refractory to cisplatin and irradiation. Since PI3K-AKT altered were dependent on exogenous glutamine, it was expected that QD cells would have lower GLS levels to inhibit glutaminolysis. However, GLS levels in WT and QD were similar. Altogether, these data indicate that in PI3K-AKT altered cervical tumor glutamine antagonists can reduce tumor proliferation. Further glutamine reduction does not alter GLS levels and hence GLS can be targeted as well to further improve treatment outcomes. To evaluate the alternate metabolic dependencies in cervical cancer tumors, both cellular as well as secreted metabolites were evaluated. To identify alternative pathways that are activated in these glutamine-deprived cell lines, we cultured these cells in uniformly labeled 13C glucose. Mass spectrometric studies also evaluated the protein levels of various metabolic intermediates in glutamine-deprived cell lines compared to control cells. To confirm our observations, we used kit-based assays to evaluate metabolites in the cells. To further confirm the change in the metabolite pathway we evaluated changes in transcriptional changes of regulating proteins/enzymes. This pre-clinical study reinforced efforts that are anticipated to reveal new metabolic vulnerabilities of cancer cells that can be targeted by therapeutic interventions. This study also helped us evaluate druggable metabolic targets that can be targeted for effective tumor eradication along with conventional treatment. This understanding will help advance the pre-clinical trials persistent to the cure of cervical cancer. Citation Format: Dr Julie K. Schwarz, Dr Nishanth Gabriel, Aunannya Banik. Evaluating strategies to harness metabolic vulnerabilities in cervical [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1114.

Prevalence of PD-L1 in Cervical Cancer Patients and the Potential for Combining an Immune Checkpoint Inhibitor With Lenvatinib.

Cervical cancer is the fourth most common cause of cancer-related deaths in women worldwide. The potential for targeted therapy against the immune checkpoint programmed death 1 (PD-1)/programmed death-ligand 1 (PD-L1) and receptor tyrosine kinases was examined in cervical cancer patients and cell lines. On tissue microarrays, PD-L1 was analyzed in 123 samples of patients with cervical cancer using immunohistochemistry. In SiHa, HeLa, and CaSki cervical cancer cell lines we examined the combination of lenvatinib with a PD-1/PD-L1 inhibitor using cell viability assays, the activation of cell signaling pathway proteins using western blots and gene expression using quantitative reverse transcriptase-PCR. Of 113 evaluable samples, 90 (79.6%) had more than 1% PD-L1 positive cells. The single treatment with the PD-1/PD-L1 inhibitor resulted in the greatest reduction in growth for CaSki and lenvatinib in HeLa cells. In contrast, the combined treatment of lenvatinib with the PD-1/PD-L1 inhibitor demonstrated a significantly stronger impeded proliferation compared to the single treatment in all three cell lines. Moreover, the combined treatment caused significantly less phosphorylation of the signaling molecules ERK and S6 in SiHa and of S6 and STAT3 in HeLa cells but not in CaSki. All treatments diminished the mRNA levels of PD-L1, Il-8, and FGFR in SiHa cells. PD1 is frequently expressed in cervical cancer samples. Combining lenvatinib with a PD-1/PD-L1 inhibitor diminished proliferation of cervical cancer cell lines. Consequently, this combination might be a promising option to treat cervical cancer. Signaling pathways involved in tumor cell growth are blocked by the combined treatment but still a model of the underlying mechanism cannot be drawn.

The CXCLs-CXCR2 axis modulates the cross-communication between tumor-associated neutrophils and tumor cells in cervical cancer

ABSTRACT Objective This study aimed to check the expression profile of the C-X-C motif chemokine ligands (CXCLs)-C-X-C motif chemokine receptor 2 (CXCR2) axis in cervical cancer and to explore the cross-talk between cervical cancer cells and neutrophils via CXCLs-CXCR2 axis. Methods Available RNA-sequencing data based on bulk tissues and single-cell/nucleus RNA-sequencing data were used for bioinformatic analysis. Cervical cancer cell lines Hela and SiHa cells were utilized for in vitro and in vivo studies. Results Except for neutrophils, CXCR2 mRNA expression is limited in other types of cells in the cervical tumor microenvironment. CXCLs bind to CXCR2 and are mainly expressed by tumor cells. CXCL1, 2, 3, 5, 6, and 8, which are consistently associated with neutrophil infiltration, are also linked to poor prognosis. SB225002 (a CXCR2 inhibitor) treatment significantly impairs SiHa cell-induced neutrophil migration. CXCL1, CXCL2, CXCL5, or CXCL8 neutralized conditioned medium from SiHa cells have weaker recruiting effects. The conditioned medium of neutrophils from healthy donors can slow cancer cell proliferation. Conditioned medium of tumor-associated neutrophils (TANs) can drastically enhance cervical cancer cell growth in vitro and in vivo. Conclusions The CXCLs-CXCR2 axis is critical in neutrophil recruitment and tumor cell proliferation in the cervical cancer microenvironment.

Synthesis, Characterization, in silico DFT, Molecular docking, ADMET Profiling Studies and Toxicity Predictions of Ag(I) Complex Derived from 4‐Aminoacetophenone

AbstractThe reaction of the synthesized (E)‐1‐(4‐((2,4‐dihydroxy‐6‐methylphenyl)diazenyl)phenyl)ethan‐1‐one, (DMPDE) ligand with Ag(I) ion at room temperature resulted in the formation of the complex; [Ag(DMPDE)(H2O)2]. 1H NMR, 13C NMR, FTIR, UV‐Vis, mass spectra, elemental analyses, thermal analysis (TGA/DTA), and molar conductance measurements were done to elucidate the structure of synthetic compounds. The results revealed an interesting geometrical variation; tetrahedral for Ag(I) complex. FT‐IR spectra demonstrated that the ligand (DMPDE) under investigation behaves as a bidentate ligand (N,O) through the nitrogen atom of the azo group which is the farthest of the acetophenone moiety and oxygen phenolic for benzene moiety and forms a stable five‐membered chelating ring. The electronic structure, molecular electrostatic potential (MEP) and quantum chemical calculations of the newly synthesized compounds are investigated theoretically at the DFT/B3LYP level of theory. Additionally, the ligand (DMPDE) and Ag(I) complex were screened against the growth of pathogenic bacteria [Actinomycosis (G+), E. Escherichia Coli (G−)] and fungi (Penicillium spp.) compared with the reference antibiotics, Chloramphenicol and Nystatin. Furthermore, 1,1‐diphenyl‐2‐picrylhydrazyl (DPPH) was used to evaluate the antioxidant activities of the ligand and its complex, which showed they both possess significant antioxidant properties in comparison with L‐ascorbic acid (Vit. C) as standard. Based on docking studies, (DMPDE) and Ag(I) complex demonstrated a greater affinity for (PDB ID: 3T88), which corresponds to Escherichia coli protein (−6.7818 kcal/mol) and (−6.7928 kcal/mol), respectively. Interestingly, the most active Ag(I) complex inside the active site of cervical cancer receptor (PDB ID: 4XR8) demonstrated a higher binding energy (−6.2631 kcal/mol) than free ligand (−5.8561 kcal/mol). In silico, ADMET displayed that compounds obey the Lipinski rule and the “Veber's rule. Consequently, is likely to exhibit oral bioavailability with good LD50 and a safety profile that includes non‐cytotoxicity, non‐immunotoxicity, and non‐skin sensitization. Finally, the compounds synthesized showed significant anticancer activity in human cervical cancer cell lines HeLa and SiHa compared with positive controls (cisplatin) and normal human hepatic cells (WRL‐68). In the present study, all tested cancer cell lines showed promising activity against Ag(I) complex, whose IC50 value ranged from (61.02 to 71.09) μg/mL.

Parthenolide inhibits the proliferation and migration of cervical cancer cells via FAK/GSK3β pathway.

Cervical cancer (CC) ranks as the fourth most prevalent malignancy among women worldwide, necessitating effective therapeutic interventions to mitigate its detrimental impact on both physical and mental health. Parthenolide (PTL), a natural product of the sesquiterpene lactone derived from Feverfew leaves, has exhibited promising anti-tumor properties in previous studies; however, its precise effects and underlying molecular mechanisms in CC remain elusive. In this work, we investigated the effect of PTL on the proliferation and migration of CC cells. Western blot analysis and Reverse transcription‑quantitative PCR were used for mechanistic elucidation. Our findings indicated that PTL substantially inhibited the proliferation of HeLa and SiHa CC cell lines in a dose- and time-dependent manner. Moreover, PTL significantly suppressed the migration of CC cells by down-regulating the expression of vascular endothelial growth factor (VEGF), metastasis-associated protein 1 (MTA1), and transforming growth factor-β1 (TGF-β1). Mechanistically, PTL blocked the phosphorylation of focal adhesion kinase (FAK) and glycogen synthase kinase-3β (GSK3β) induced by epidermal growth factor (EGF). Further investigations revealed that PTL suppressed the proliferation of CC cells by inhibiting the EGF-mediated phosphorylation of the FAK/GSK3β signaling pathway. Taken together, the present in vitro results suggest that PTL may inhibit the proliferation and migration of CC cells through down-regulating the FAK/GSK3β signaling pathway, providing new insights for the application of PTL in the treatment of CC.

Maackiain suppresses the development of cervical cancer via AMPK priming autophagy.

Maackiain (Mac), a flavonoid analog isolated from Sophora flavescens, exhibits neuroprotective, anti-allergic, anti-inflammatory, and pro-apoptotic effects. It is not clear whether Mac has a therapeutic effect on cervical cancer. In this work, we used RT-qPCR, western blot, immunofluorescence, and related methods to detect the therapeutic mechanism of Mac for cervical cancer. We demonstrated that Mac significantly inhibited the proliferation, migration, and invasion of human cervical cancer cell lines HeLa and SiHa. And, Mac enhanced the pro-apoptotic effects of cisplatin in treating cervical cancer cells. Mac has shown good efficacy in treating cervical cancer. Furthermore, Mac inhibited the mammalian target of the rapamycin (mTOR) pathway, thereby inducing autophagy in cervical cancer cells. The regulation of mTOR/autophagy pathway by Mac relied on the activation of AMP-activated protein kinase (AMPK), and the inhibition of the AMPK reversed the Mac's anti-cervical cancer activity. In addition, experimental study of Mac in mouse xenograft tumor model further confirmed its good anti-cervical cancer activity. Mac inhibits human cervical cancer by activating the AMPK/mTOR/autophagy pathway, indicating that it is a potential natural compound for the treatment of cervical cancer. This study also provides a feasible molecular mechanism for the treatment of cervical cancer.

Effects of the PARP Inhibitor Niraparib on the Radiosensitivity of Human Lung and Cervical Cancer Cells

DNA damage repair (DDR) initiated after DNA damage may lead to a decreased radiosensitivity of tumor cells or even radiation resistance. Previous studies indicate that PARP inhibitors block the repair of DNA single-strand breaks (SSBs), and unrepaired SSBs may continuously accumulate and form double-strand breaks (DSBs) that promotes tumor cell death. Studies have also shown that PARP inhibitors act synergistically with RT to inhibit DDR. In this study, we aim to investigate the effect of Niraparib, a clinically approved PARP inhibitor on the radiosensitivity of human lung and cervical cancer cells, and preliminarily explore the underlying mechanism. The human lung cancer cell line A549 and human cervical cancer cell line Siha were both treated with niraparib (for 1h), or X-ray (4Gy), or Niraparib (for 1h) combined with X-ray (4Gy). The effect was examined via measurements of cell proliferation by the cell counting kit-8 assay, and cell viability was detected by clone formation assays. Cell apoptosis and cell cycle distribution were investigated by flow cytometry. In human lung and cervical cancer cell lines, Niraparib combined with radiation therapy significantly inhibited cell proliferation. The proportion of apoptotic cells in cell lines treated with Niraparib plus radiation (the combination group) was significantly higher compared with control, radiation-only, and Niraparib-only groups. Additionally, the proportion of A549 cells in the G2/M phase was significantly increased in the combination group compared with the radiation-only group. The results support that PARP inhibitor Niraparib increases the radiosensitivity of tumor cells, promotes their apoptosis, and induces cell cycle redistribution. The possible mechanism is associated with the inhibition of radiation-induced DNA damage repair.

Preparation and characterization of ionic metal-based anticancer formulations of the type [bis-DACH tolfenamate metal {Co(II), Cu(II) and Zn(II)}] complexes: Binding studies with ct-DNA/tRNA, cleavage and cytotoxic activity against chemoresistant cancer cells

A series of ionic metallo-NSAID’s having the formulae [Co(DACH)2(H2O)2](tolf)21, [Cu(DACH)2(H2O)2](tolf)22 and [Zn(DACH)2(H2O)2](tolf)23 where DACH and tolf− are 1,2-diaminocyclohexane and tolfenamate anion, respectively were synthesized. Complexes 1–3 were characterized by FT–IR, EPR, 1H/13C NMR, UV–vis and ESI–MS spectrometry methods. The bonding features as well as the electronic structure of all the complexes were optimized using DFT studies. Binding affinity of parent NSAID and 1–3 complexes with ct–DNA/tRNA were studied by using various complementary biophysical methods. The corroborative results of these experimental studies revealed that they were capable to interact with the therapeutic targets, ct–DNA/tRNA by the electrostatic interactions, showing higher preference for tRNA as compared to ct–DNA as quantified by their binding strength (Kb and Ksv values). It was also observed that the complexes exhibited much higher binding affinity than the parent free NSAID. A concentration-dependent cleavage of the complexes was observed with tRNA which promoted the hydrolysis of RNA. The cytotoxicity of 1–3 was evaluated against chemo-resistant phenotypes of cancers, viz., breast cancer cell line (MCF- 7), cervical cancer cell line (SiHa), triple-negative breast cancer cell (MDA-MB-231), oral squamous cancer cell (AW13516) and lung cancer cell line (Hop-62) by SRB assay. The cytotoxicity activity results displayed that among all the ionic tolfenamate metal complexes, complex 2 showed a much superior cytotoxic profile against treated cancer cell lines (SiHa, MDA-MB-231, AW13516, MCF-7 and Hop-62) with a good GI50 value of (4.7 ± 0.03) µg/ml, (10.5 ± 0.36) µg/ml, (19.2 ± 0.02) µg/ml, (4.1 ± 0.05) µg/ml, and (18.2 ± 0.04) µg/ml, respectively, while the complexes 1 and 3 showed moderate activity. Cell morphology studies were carried out in AW13516, MDA–MB–231, MCF–7 and Hop–62 cells with the incubation of complexes for a period of 48 h which implicated that the cells exposed to complex 2 presented round features and were nonadherent in culture. These morphological changes indicated that complex 2 was capable of inducing cell death, probably by apoptosis suggesting copper complex 2 was promising potent anticancer agent.

WIF1 was downregulated in cervical cancer due to promoter methylation.

Wnt inhibitory factor 1 (WIF1) is frequently downregulated in a variety of cancer due to promoter methylation. However, the methylation status of the WIF1 promoter in cervical cancer remains unclear. This study aimed to elucidate the mechanism by which WIF1 promoter methylation contributes to cervical cancer development. The expression of WIF1 in cervical cancer tissues was examined by immunohistochemistry. The methylation status of the WIF1 promoter in cervical cancer cells was detected by methylation specific PCR. WIF1 mRNA levels and protein levels were detected by PCR and Western blot analysis. We found that WIF1 expression was low in cervical cancer tissues compared to adjacent normal cervical tissues. The WIF1 promoter was methylated in the cervical cancer SiHa cell line but not in the normal cervical epithelial cell line Ect1. Correspondingly, WIF1 mRNA levels and protein levels were significantly lower in SiHa cells than in Ect1 cells. Treatment with 5-aza-2-deoxycytidine (AZA) led to the upregulation of WIF1 mRNA and protein levels in SiHa cells, but the effects were abrogated by treatment with WIF1 siRNA. In addition, AZA treatment induced apoptosis and inhibited the invasion of SiHa cells, and the effects were abrogated by WIF1 siRNA. The protein levels of survivin, c-myc and cyclinD1 were significantly lower in SiHa cells treated with AZA, but their levels were upregulated after treatment with WIF1 siRNA. In conclusion, the methylation of the WIF1 promoter leads to the downregulation of WIF1 and the activation of Wnt/β-catenin signaling in cervical cancer cells. WIF1 is a tumor suppressor that is inactivated in cervical cancer.

N-Acetyl-L-Cysteine Reduces Cervical Carcinogenesis by Promoting Apoptosis.

Cervical cancer is the fourth leading cause of cancer death in women, and is one of the most common malignant tumors of the reproductive system. However, more effective treatment for cervical cancer is needed. In this study, we aim to investigate whether N-acetyl-L-cysteine (NAC) could inhibit the proliferation of human papillomavirus (HPV)-positive cells, and reduce cervical carcinogenesis. The cervical cancer cell lines SiHa, HeLa, HPV-negative cell line C33A, and the immortalized human cervical keratinocyte cells S12 were used. The protein expression was determined using Western blot assay. mRNA expression was determined using quantitative reverse transcription polymerase chain reaction (qRT-PCR). Cell proliferation was determined by Cell Counting Kit-8 assay. Cell apoptosis was evaluated using Annexin V-FITC apoptosis kits. The numbers of colonies were measured using colony-forming assay. Xenograft tumor necrosis and HPV16 E7 expression were determined using hematoxylin and eosin (H&E) staining and immunohistochemistry. Our results showed that NAC treatment at the concentration of 1.5mM significantly promoted cell apoptosis and reduced cell growth by inhibiting HPV16 E7 expression. NAC inhibited HPV16-oncoprotein-induced hypoxia-inducible factor (HIF)-1α protein expression and Akt activation invitro. Additionally, NAC suppressed tumor growth, as evidenced by the smaller tumor size in the xenograft mouse model and decreased HPV16 E7 expression in tumor tissues. Our findings demonstrate that NAC exhibits the potential to promote HPV-positive cell apoptosis, and suppress the proliferation of HPV-positive cells by inhibiting cell inhibitor of apoptosis protein 2 and HIF-1α.

Effects of the PARP inhibitor Niraparib on the radiosensitivity of human lung and cervical cancer cells

ObjectiveTo investigate the effect of Niraparib, a clinically approved PARP inhibitor on the radiosensitivity of human lung and cervical cancer cells, and preliminarily explore the underlying mechanism. MethodsThe human lung cancer cell line A549 and human cervical cancer cell line Siha were both treated with Niraparib for 1 ​h, or X-ray irradiation with 4 ​Gy, or Niraparib for 1 ​h combined with X-ray irradiation with 4 ​Gy. The effect was examined via measurements of cell proliferation by the cell counting kit-8 assay, and cell viability was detected by clone formation assays. Cell apoptosis and cell cycle distribution were investigated by flow cytometry. ResultsIn human lung and cervical cancer cell lines, Niraparib combined with radiation therapy significantly inhibited cell proliferation. The proportion of apoptotic cells in cell lines treated with Niraparib plus radiation (the combination group) was significantly higher compared with control, radiation, and Niraparib groups (P ​< ​0.05). Additionally, the proportion of A549 ​cells in the G2/M phase was significantly increased in the combination group compared with the radiation group (P ​< ​0.05). ConclusionPARP inhibitor Niraparib increases the radiosensitivity of tumorcells, promotes their apoptosis, and induces cell cycle redistribution. The possible mechanism is associated with the inhibition of radiation-induced DNA damage repair.

Combined GSK-3β and MEK inhibitors modulate the stemness and radiotherapy sensitivity of cervical cancer stem cells through the Wnt signaling pathway

Cancer stem cells (CSCs) are the basis of cancer and lead to the recurrence and metastasis of cervical cancer. The aim of this study was to investigate the effects of antineoplastic agents on the stemness and radiotherapy sensitivity of cervical CSCs. Side population (SP) and non-side population (NSP) cells from the SiHa cervical cancer cell line were separated using flow cytometry. The cell spheroidization, proliferation, and subcutaneous tumor formation abilities of SP cells were stronger than those of NSP cells, and cervical CSC marker expressions increased in SP cells. The proliferation, anti-apoptosis and migration of SP cells under ionizing radiation were higher than those of SiHa cells. GSK-3β and/or MEK inhibitors can increase the proliferation, migration and anti-apoptosis of SP cells, and CSC marker expressions. The Wnt pathway inhibitor decreased CSC stemness maintenance by combination of GSK-3β and MEK inhibitors. Injection of GSK-3β and MEK inhibitors under ionizing radiation promoted tumor growth and activated downstream factor expressions in the Wnt signaling pathway in vivo. This study demonstrated that combining GSK-3β and MEK inhibitors can activate Wnt signaling pathway in cervical CSCs, thereby affecting their stemness maintenance and radiotherapy sensitivity.

Iron oxide-silver-curcumin nanocomposite acts against HPV16 positive cervical cancer cell siha by triggering crosstalk between autophagy and apoptosis primarily via breach in cellular redox equilibrium

Curcumin, the dietary phytocompound obtained from Curcuma sp. possesses multiple bioactivities of which its appreciable anti-tumour activity has been evidenced pertaining to cytotoxicity mediated through different cell death pathways. However, hydrophobicity of this molecule poses a major hindrance as it directly affects bioavailability and thus anti-tumor property. Currently, cervical cancer is one of the most commonly occurring women cancers worldwide and is a growing menace especially in the developing countries. In this study we have tried to formulate a novel green-synthesised iron oxide-silver nanocomposite-based delivery system for curcumin so as to improve its druggability against the HPV-16 positive cervical cancer cell line SiHa. Different microscopic, flow cytometric, spectrophotometric and immunoblotting assays were used to analyse its effects on the targeted cells. We have observed that the curcumin-nanocomposite showed enhanced anti-proliferative capability compared to free curcumin against SiHa showing increased cellular uptake at 24 h treatment. Further, it modulated the PI3K/AKT/mTOR signalling cascade to induce both autophagic and apoptotic death in the target cells. Such changes were mainly orchestrated through imbalance in intracellular redox system and resultant augmented ROS accumulation. Our study suggests that this bimetallic nanocomposite can aid in increasing bioavailability and concomitant therapeutic efficacy of curcumin.

2-Deoxy-D-glucose simultaneously targets glycolysis and Wnt/β-catenin signaling to inhibit cervical cancer progression.

Cervical cancer is one of the most common female malignant tumors, with typical cancer metabolism characteristics of increased glycolysis flux and lactate accumulation. 2-Deoxy-D-glucose (2-DG) is a glycolysis inhibitor that acts on hexokinase, the first rate-limiting enzyme in the glycolysis pathway. In this research, we demonstrated that 2-DG effectively reduced glycolysis and impaired mitochondrial function in cervical cancer cell lines HeLa and SiHa. Cell function experiments revealed that 2-DG significantly inhibited cell growth, migration, and invasion, and induced G0/G1 phase arrest at non-cytotoxic concentrations. In addition, we found that 2-DG down-regulated Wingless-type (Wnt)/β-catenin signaling. Mechanistically, 2-DG accelerated the degradation of β-catenin protein, which resulted in the decrease of β-catenin expression in both nucleus and cytoplasm. The Wnt agonist lithium chloride and β-catenin overexpression vector could partially reverse the inhibition of malignant phenotype by 2-DG. These data suggested that 2-DG exerted its anti-cancer effects on cervical cancer by co-targeting glycolysis and Wnt/β-catenin signaling. As expected, the combination of 2-DG and Wnt inhibitor synergistically inhibited cell growth. It is noteworthy that, down-regulation of Wnt/β-catenin signaling also inhibited glycolysis, indicating a similar positive feedback regulation between glycolysis and Wnt/β-catenin signaling. In conclusion, we investigated the molecular mechanism by which 2-DG inhibits the progression of cervical cancer in vitro, elucidated the interregulation between glycolysis and Wnt/β-catenin signaling, and preliminarily explored the effect of combined targeting of glycolysis and Wnt/β-catenin signaling on cell proliferation, which provides more possibilities for the formulation of subsequent clinical treatment strategies.

Hypoxia-induced paclitaxel resistance in cervical cancer modulated by miR-100 targeting of USP15

ObjectiveHypoxia, which occurs during the development of cervical cancer, confers chemotherapy resistance. MicroRNA expression is regulated by hypoxia and is associated with the onset and progression of certain types of cancer. MicroRNA-100 (miR-100) is a microRNA, associated with nasopharyngeal and oral squamous cell carcinomas, whose expression is decreased in cervical cancer. This study aims to ascertain the effect of hypoxia on expression levels of both miR-100 and its target genes, as well as exploring the sensitivity to paclitaxel under hypoxic conditions. MethodsWe investigated the effect of hypoxia on miR-100 expression. We also explored the regulators of paclitaxel response under hypoxic conditions of cervical cancer. ResultsUsing RT-qPCR, we found that expression of miR-100 in cervical cancer cell lines SiHa and HeLa is significantly higher under hypoxic conditions (1% O2). We also confirmed that human ubiquitin-specific protease 15 (USP15) is the one of the target proteins of miR-100. Hypoxia and overexpression of miR-100 both reduced the activity of the luciferase reporter containing the 3′-untranslated region of USP15, which contains the miR-100 binding site. Furthermore, a western blot analysis showed that hypoxia suppresses the expression of the USP15 protein, while RT-qPCR showed hypoxia-induced downregulation of USP15 mRNA levels. We also discovered that overexpression of miR-100 induces paclitaxel resistance, thereby reducing the drug’s therapeutic effect on cell death. ConclusionsOur results are consistent with the hypothesis that cervical cancer cells overexpress miR-100 in response to hypoxia and that miR-100 is a facilitator of USP15 downregulation and inactivation.

Fe(II) complexes of 2,2':6',2''-terpyridine ligands functionalized with substituted-phenyl groups: synthesis, crystal structures and anticancer potential.

With the aim of developing potential anticancer drug candidates, a series of Fe(II) complexes were synthesized using nine 2,2':6',2''-terpyridine ligands functionalized with substituted-phenyl groups, and their biological activities were systematically investigated. Their bis-terpyridine sandwich-like structures were determined by single crystal X-ray crystallography. In vitro antiproliferative experiments based on three human cancer cell lines, including human hepatoma cancer cell line (Bel-7402), human esophageal cancer cell line (Eca-109), and human cervical squamous cancer cell line (SiHa), indicate the high antiproliferation activities of these complexes compared with commercial cisplatin. And their toxicity to normal cells was estimated based on human normal hepatocyte (HL-7702) cell line. In particular, when the phenyl in terpyridine ligand was modified by a carboxyl group, the corresponding complex 3 exhibited much higher antiproliferation to cancer Bel-7402 cells (IC50 = 3.653 μmol L-1) than cisplatin and low toxicity to normal HL-7702 cells (IC50 = 99.92 μmol L-1), implying a significant selectivity for 3 in killing hepatoma cancer cells. Combined with the fact that iron element is more accessible than platin, this series of Fe(II) complexes comprises potential candidates for anticancer drugs with specific inhibition of hepatoma cancer. UV titration experiments and circular dichroism (CD) showed a strong binding affinity between these nine complexes and CT-DNA. However, molecular docking simulation revealed the competitive binding of DNA and protein to these complexes. Further, the interactions between these complexes and bovine serum albumin (BSA) have been studied by fluorescence titration and CD spectroscopy.